Difference detection methods using matched multiple dyes

ABSTRACT

A process and a kit are provided for detecting differences in two or more samples of protein, including proteins bearing post-translational modifications and peptides. Proteins are prepared, for example, from each of a different group of cell samples or body fluid samples to be compared. Each protein extract is labeled with a different one of a luminescent dye from a matched set of dyes. The matched dyes have generally the same ionic and pH characteristics but emit light at different wavelengths to exhibit a different color upon luminescence detection. The labeled protein extracts are mixed together and separated together by electrophoresis or a chromatographic method. The separation is observed to detect proteins unique to one sample or present in a greater ratio in one sample than in the other. Those unique or excess proteins will fluoresce the color of one of the dyes used. Proteins common to each sample migrate together and fluoresce the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 08/425,480,filed Apr. 20, 1995, now U.S. Pat. No. 6,127,134.

BACKGROUND OF THE INVENTION

The present invention relates to a process for detecting differences inprotein compositions, including proteins bearing post-translationalmodifications, and more particularly, to a process utilizing a matchedpair of labeling reagents for detecting such differences.

Researchers studying various aspects of cell biology use a variety oftools to detect and monitor differences in cell structure, function anddevelopment. An essential part of studying cells is studying thedifferences and similarities in the protein composition between thedifferent cell types, stages of development and condition. Determiningdifferences in the protein content between normal and cancerous cells orwild type and mutant cells, for example, can be a valuable source ofinformation and a valuable diagnostic tool.

Mixtures of proteins can be separated into individual components byvarious means, including electrophoresis and chromatography. Separationaccording to differences in mass can be achieved by electrophoresing ina polyacrylamide gel under denaturing conditions. One dimensional andtwo dimensional gel electrophoresis have become standard tools forstudying proteins. One dimensional SDS (sodium dodecyl sulfate)electrophoresis through a cylindrical or slab gel reveals only the majorproteins present in a sample tested. Two dimensional polyacrylamide gelelectrophoresis (2D PAGE), which separates proteins by isoelectricfocusing, i.e., by charge in one dimension and by size in the seconddimension, is the more sensitive method of separation and will provideresolution of most of the proteins in a sample.

The proteins migrate in one- or two-dimensional gels as bands or spots,respectively. The separated proteins are visualized by a variety ofmethods; by staining with a protein specific dye, by protein mediatedsilver precipitation, autoradiographic detection of radioactivelylabeled protein, and by covalent attachment of fluorescent compounds.The latter method has been heretofore only able to be performed afterthe isoelectric focusing step of 2D PAGE. Immediately following theelectrophoresis, the resulting gel patterns may be visualized by eye,photographically or by electronic image capture, for example, by using acooled charge-coupled device (CCD).

To compare samples of proteins from different sources, such as differentcells or different stages of cell development by conventional methods,each different sample is presently run on separate lanes of a onedimensional gel or separate two dimensional gels. Comparison is byvisual examination or electronic imaging, for example, by computer-aidedimage analysis of digitized one or two dimensional gels.

Two dimensional electrophoresis is frequently used by researchers.O'Farrell, P. H., “High resolution two-dimensional electrophoresis ofproteins”, Journal of Biological Chemistry, 250:4007-4021 (1975),separated proteins according to their respective isoelectric points inthe first dimension by the now well known technique of isoelectricfocusing and by molecular weight in the second dimension bydiscontinuous SDS electrophoresis. Garrels, J. I., “Two-dimensional GelElectrophoresis and Computer Analysis of Proteins Synthesized By ClonalCell Lines”, Journal of Biological Chemistry, Vol. 254, No. 16,7961-7977 (1979), used a two dimensional gel electrophoresis system tostudy the pattern of protein synthesis in nerve cells and glial cells.Garrels conducted a comparative analysis of data from multiple samplesto correlate the presence of particular proteins with specificfunctions. Computerized scanning equipment was used to scan a section ofthe gel fluorogram, detect the spots and integrate their densities. Theinformation was stored and plotted according to intensity in each ofseveral different scans.

Urwin, V. E. and Jackson, P., “A multiple High-resolution MiniTwo-dimensional Polyacrylamide Gel Electrophoresis System: ImagingTwo-dimensional Gels Using A Cooled Charge-Coupled Device After StainingWith Silver Or Labeling With Fluorophore”, Analytical Biochemistry195:30-37 (1991) describes a technique wherein several isoelectricfocusing (IEF) gels were used to separate proteins by charge, thenloaded onto a gradient slab gel such that the IEF gels were positionedend to end along the top of the slab gel. The gels were thenelectrophoresed. The resulting protein spots were visualized either bystaining the second dimensional slab gel with silver or by fluorescentlabeling following the isoelectric focusing step. Labeling must takeplace after the first electrophoresis, i.e., the isoelectric focusingbecause the presence of the fluorescein label on the protein changes theisoelectric point of the protein when subjected to electrophoresis. Inaddition, the label attaches to a sulfur on the protein forming anunstable bond which would tend to break during isoelectric focusing ifthe label is attached prior to the electrophoresis step. An article bySantaren, J. et al., “Identification of Drosophila Wing Imaginal DiscProteins by Two-Dimensional Gel Analysis and Microsequencing”,Experimental Cell Research 206: 220-226 (1993), describes the use ofhigh resolution two dimensional gel electrophoresis to identify proteinsin Drosophila melanogaster. The dry gel was exposed to X-ray film forfive days. The developed X-ray film is analyzed by a computer todetermine the differences in the samples.

Two dimensional gel electrophoresis has been a powerful tool forresolving complex mixtures of proteins. The differences between theproteins, however, can be subtle. Imperfections in the gel can interferewith accurate observations. In order to minimize the imperfections, thegels provided in commercially available electrophoresis systems areprepared with exacting precision. Even with-meticulous controls, no twogels are identical. The gels may differ one from the other in pHgradients or uniformity. In addition, the electrophoresis conditionsfrom one run to the next may be different. Computer software has beendeveloped for automated alignment of different gels. However, all of thesoftware packages are based on linear expansion or contraction of one orboth of the dimensions on two dimensional gels. The software cannotadjust for local distortions in the gels.

Protein samples may also be separated by alternative electrophoretic orchromatography techniques. Such techniques are capable ofhigh-resolution separation of proteins or peptides particularly inorthogonal combinations. However, current chromatographic systems tendto have lower resolving power than electrophoretic systems, ie thenumber of proteins or peptides capable of being separated is smaller.Typical elution traces can be found in manufacturers' catalogues, e.g.,Amersham Pharmacia Biotech “BioDirectory '99” catalogue under“Chromatography columns and media” starting at page 502. Nevertheless,chromatographic systems do have certain advantages over electrophoresisfor some applications. For example, they are often easier to automateand it is usually easier to obtain samples of the proteins followingseparation.

For these reasons, separation by chromatographic systems for proteomeprofiling for example, is of interest. For example, Opiteck andcolleagues have published examples of two-dimensional chromatographicsystems where fractions eluted from a chromatographic separation systemare applied to a second chromatographic system. (See specifically,Opiteck, Lewis and Jorgenson, Anal. Chem, vol. 69, 1518, (1997) whichdescribes the use of a cation exchange system in combination with areverse phase chromatographic system, and Opiteck et al., Anal Biochem.,vol. 258, 349, (1998), which describes the use of size exclusionchromatography in combination with reverse phase chromatography.) Theparticularly low resolving power of size exclusion chromatography isalleviated in the latter paper by using 8 size exclusion columns inseries prior to further fractionation of the eluent by reverse phasechromatography. A theoretical resolving power of 800 proteins wasestimated for this system. The limited resolving power of certainchromatographic and electrophoretic systems can also be overcome at theanalysis stage. Mass spectrometry is becoming widely used for proteinidentification following chromatographic or electrophoretic separationand can itself be used as a separation method based on mass. Forexample, Jensen et al., Anal. Chem. Vol. 71, 2076, (1999) describes theuse of capillary isoelectric focusing as a separation method and thenuses electrospray ionisation Fourier transform ion cyclotron resonancemass spectrometry to further separate proteins in the eluent from theisoelectric focusing system, as well as provide a means ofidentification.

The object of the present invention is to eliminate the problemsassociated with gel distortions or column variability and to provide asimple, relatively fast and reliable method of comparing and contrastingthe protein content of different samples.

BRIEF SUMMARY OF THE INVENTION

The foregoing objects have been achieved by the process of the presentinvention wherein differences, if any, between multiple samples ofproteins, for example, those extracted from different cells or obtainedfrom other sources, are detected by labeling each sample of suchproteins with a different one of a set of matched luminescent dyes.Proteins, as used herein, includes proteins bearing post translationalmodifications and portions thereof, including peptides. The matched dyeshave generally the same ionic and pH characteristics but absorb and/orfluoresce light at different wavelengths, producing a different colorfluorescence. In addition, the dyes should be similar in size. After anincubation period sufficient to permit the formation of covalent bondsbetween the dye and one or more attachment sites on the proteins, thelabeled samples are then mixed together and the proteins separated in asingle separation process. Separation may be by electrophoresis or bychromatographic methods. When separation is by electrophoresis on asingle gel, the proteins common to each sample co-migrate to the sameposition. Similarly, when separation is by chromatographic means in acolumn, for example, the proteins common to each sample migrate to thesame position. Proteins which are different will migrate alone todifferent locations on the gel or at different times from the column andwill fluoresce different colors, thereby identifying which initialsample has one or more proteins which differ from the other initialsample or samples.

The invention also includes a kit for performing the method of thepresent invention. The kit includes the matched set of dyes, and mayalso include materials for separating the proteins. Quench materials forstopping the reaction between the protein and the dye when necessary,may optionally be provided. Those materials may comprise, for example,electrophoresis gels or chromatography columns.

DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings are provided to the Patent andTrademark Office with payment of the necessary fee.

FIG. 1 is a schematic diagram of the process of the present invention.

FIGS. 2a) and 2 b) are images of proteins labeled with a preferredmatched pair of labels of the present invention run on a single SDSpolyacrylamide gel.

FIGS. 3a)-d) are images of portions of a two dimensional gel loaded withtwo different samples of bacterial extract, one IPTG-induced and theother uninduced, labeled with a different one of the dyes of the matchedpair of dyes according to the process of the present invention.

FIGS. 4a)-d) are images of portions of a two dimensional gel loaded withtwo different samples of bacterial extract, one having exogenously addedcarbonic anhydrase and one without carbonic anhydrase, each labeled witha different one of the dyes of the matched pair of dyes according to theprocess of the present invention.

FIGS. 5a) and b) are images of Cy2-NHS and Cy3 hydrazide labeledproteins run on SDS-PAGE, demonstrating that the hydrazide dyesspecifically label the carbohydrate portion of the glycoprotein.

FIGS. 6a) and b) are images of Cy3 and Cy5 hydrazide labeled proteinmixes on SDS-PAGE.

FIGS. 7a) and b) are images of a section of a 2DE gel loaded with Cy3hydrazide labeled HBL100 cell extract and Cy5 hydrazide labeled BT474cell extract.

FIGS. 8a) and b) are images of a 2DE gel loaded with Cy3 hydrazidelabeled BT474 cell extract without exogenously added protein and Cy5hydrazide labeled BT474 cell extract with exogenously added protein.

FIG. 9 is a graph showing the results of ion exchange chromatography ofCy3 labeled bovine serum albumin (BSA;33 μg) and Cy5 labeled transferrin(33 μg).

FIG. 10 is a graph showing the results of ion exchange chromatography ofCy5 labeled myogloblin, transferrin and bovine serum albumin (13 μg ofeach protein).

FIG. 11 is a graph showing the results of reverse phase chromatographyof Cy5 labeled ribonuclease a, cytochrome c, holo-transferrin andapomyoglobin (3.3 μg of each protein).

FIG. 12 is a graph showing the results of size exclusion chromatographyof Cy3 labeled thryoglobulin, apoferritin, IgG and β-lactoglobulin (23.5μg of each protein).

FIG. 13 is a graph showing the results of differential protein analysisby size exclusion chromatography using samples labeled with Cy3 or Cy5.The Cy3 labeled protein are thryoglobulin (38 μg), apoferritin (28 μg),IgG (2.6 μg),and β-lactoglobulin (12 μg). The Cy 5 labeled sample doesnot contain IgG. Cy3 and Cy5 labeled samples were mixed prior tochromatography.

FIG. 14 is a graph showing the results of differential analysis by ionexchange chromatography, of Cy3 or Cy5 labeled samples. Cy3 labeledsample contained transferrin and Cy5 labeled sample containedtransferrin and bovine serum albumin (13 μg of each protein). Cy3 andCy5 labeled samples were mixed prior to chromatography.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The process of the present invention employs a matched set of dyeswherein each dye in the set is generally equal to the other dyes inionic and pH characteristics, and chemical reactivity for covalentattachment to proteins, yet fluoresces at a different wavelength,thereby exhibiting a different color luminescence when viewed. The dyesare preferably roughly equal in molecular weight, but need not be. Eachone of the dyes within the matched set of dyes is used to label proteinsin a different one of a set of different samples of proteins so thateach sample is labeled with a different dye within the set of dyes.After labeling, the proteins are mixed and separated in the same mediumby any suitable known separation technique, such as electrophoresis orchromatography. Electrophoresis techniques include one or twodimensional electrophoresis, capillary zone electrophoresis, capillarygel electrophoresis, isoelectric focussing, isotacophoresis, andmicellar electrokinetic chromatography. Chromatographic techniquesinclude affinity chromatography, size exclusion chromatography, reversephase chromatography, hydrophobic interaction chromatography and ionexchange chromatography.

With reference to the schematic diagram of FIG. 1, a first extract ofproteins is prepared by known techniques from a first group of cells,then labeled with the first dye of a matched pair of dyes. A secondextract of proteins is prepared by known techniques from a second groupof cells then labeled with the second dye of the matched pair of dyes.To label the protein, the reactive form of the dye and the protein areincubated for a period of time sufficient to allow for the formation ofa covalent bond between the reactive form of the dye and potentialattachment or binding sites on the proteins. The period of time isgenerally from 15 to 30 minutes, depending on the temperature. Thetemperature range is generally from about 0° C. to 25° C. The reactionbetween the dye and the proteins may be quenched after a sufficientpercentage of available binding sites on the protein molecule arecovalently bound to the dye. Any suitable known quenching material maybe used. Other methods for removal of excess dye, such as gelfiltration, may also be used. In situations where the labelling reactionis allowed to go to completion, for example when all the reactive dyehas been used, quenching or removal of excess dye may not be required.

The first and second group of cells can be any two sets of cells theprotein content of which one wishes to compare or contrast. For example,the first group of cells can be the wild-type, or normal, cells, and thesecond group of cells can be mutant cells from the same species.Alternatively, the first group of cells can be normal cells and thesecond group can be cancerous cells from the same individual. Cells fromthe same individual at different stages of development or differentphases of the cell cycle can be used also. The cells from a developingembryo, from the ventral furrow of Drosophila melanogaster, for example,can be harvested as the first group of cells and cells that developadjacent to the ventral furrow cells can be harvested as the secondgroup of cells. The differences in protein composition between cells ofthe same type from different species can also be the subject of study bythe process of the present invention. In addition, the process of thepresent invention can be used to monitor how cells respond to a varietyof stimuli or drugs. All of the events that might alter cell behavior asexpressed through protein changes can be detected without the need andexpense of high precision 2D PAGE systems. Those skilled in the art willrecognize that the proteins for comparison may also be derived frombiological fluids, such as serum, urine, or spinal fluid.

The labeled samples are mixed and, as illustrated in FIG. 1, applied inmeasured aliquots to one gel, then preferably subjected to 2D PAGE. Onedimensional SDS electrophoresis can be used instead of 2D PAGE. Theprocedures for running one dimensional and two dimensionalelectrophoresis are well known to those skilled in the art.

Proteins that the two cell groups have in common form coincident spots.The ratio of the fluorescent intensity between identical proteins fromeither group will be constant for the vast majority of proteins.Proteins that the two groups do not have in common will migrateindependently. Thus, a protein that is unique or of different relativeconcentration to one group will have a different ratio of fluorescenceintensity from the majority of protein spots, and will produce a colorspecific for one or the other of the protein extracts, depending on thelabel used. For example, the proteins that are in the first sample maybe labeled red, while the second group is labeled blue. Under conditionswhere exactly equal amounts of protein from each group is mixed togetherand run on the same gel the ratio of fluorescence intensity will be onefor the majority of proteins. Those proteins that are distinct to one orthe other group will have a fluorescence intensity ratio less than orgreater than one, depending on the order or ratioing.

The gel can be analyzed by a two wavelength fluorescence scanner, by afluorescent microscope or by any known means for detecting fluorescence.Gel analysis can be completely automated by means of computer aidedidentification of protein differences. Using an electronic detectionsystem such as a laser scanning system with a photo multiplier tube or acharged-coupled device (CCD) camera and a white light source, twoelectronic images are made of the wet gel using different known filtersets to accommodate the different spectral characteristics of thelabels. One image views fluorescence of the first dye using a firstfilter appropriate to filter out all light except that emitted at thewavelength of the first dye and the other image, views fluorescence ofthe second dye using a second filter, appropriate to filter out alllight except that emitted at the wavelength of the second dye. Exposureis about 5 to 500 seconds. The differences in the samples can beidentified, either during electrophoresis or in less than ½ hourfollowing electrophoresis. Several software packages are commerciallyavailable which will either subtract the first image from the second toidentify spots that are different, or, alternatively, the images may bedivided to leave only the spots not common to both images. Insubtracting the images, like spots will cancel each other, leaving onlythose that are different. In ratio analysis, like spots will provide avalue of one. Differences will result in values greater, than one lessthan one.

In conventional analysis, a control is run with known proteins for thecell type being studied. The known spots on the sample gel have to beidentified and marked, compared to the control and the second gel todetermine differences between the two gels. In the present invention,there is only one gel so no marking is necessary. In addition, thesoftware used on conventional processes for alignment of different gelsprior to comparing and contrasting protein differences does not correctfor local distortions and inconsistencies between two or more gels. Theprocess of the present invention eliminates the need for such correctionbecause the labeled proteins for all samples to be tested are mixed andseparated together. Any distortions in an electrophoresis gel, forexample, are experienced equally by each sample.

Selection and synthesis of the matched set of dyes is important. In theprocess of the present invention, the fluorescent dyes are covalentlycoupled to proteins, preferably via lysine residues of the proteins, butcoupling may also be to sulfhydryl or carboxylic acid groups in theproteins. For modified proteins, the dyes may be coupled to themodifying groups; for example the dyes may be coupled to the sugarresidue of glycoproteins following oxidation thereof to the aldehyde.Regulation of the pH of proteins to force attachment of labels at oneamino acid residue to the exclusion of other amino acids is a well knowntechnique, as set forth in R. Baker, Organic Chemistry of BiologicalComponents, (Prentice Hall, pub. 1971). For analysis of proteins, aplurality of attachment sites are labeled. The optimum percentage ofattachment sites labeled will depend on the dyes and target functionalgroups chosen. When the preferred dyes specifically discussedhereinbelow are used to label lysines; preferably no more than 2% of theattachment sites and more preferably, slightly less than 1%, arelabeled, to avoid rendering the protein insoluble. Thus, where a typicalprotein is composed of about 7% lysines, there will be less than onemodified amino acid per one thousand. A typical protein is composed ofabout 450 amino acids. An alternative strategy is to label all thefunctional groups of a particular type which is less prevalent in theprotein, for example sulfhydryl groups in cysteines. When lysine is theattachment site, the covalent linkage destroys the positive charge ofthe primary amine of the lysine. Because isoelectric focusing depends oncharge, it is important to compensate for the charge loss. A basicresidue should remain basic. Changing the pKa of one residue per proteinby as much as 3 can be tolerated, provided the basicity or acidity ofthe modified residue, as the case may be, is not altered. Dyes likerhodamine and fluorescein are not suitable because of the difference incharge.

Those skilled in the art will recognize that the labeling approachesdescribed could equally well be applied to peptide molecules derivedfrom cells or present in biological fluids such as plasma, serum, urine,ascites or spinal fluid. Furthermore, in preparing a protein sample forlabeling it may, in certain circumstances, be beneficial to firstperform an enzyme digestion with trypsin or other protease enzymes togenerate peptides prior to labeling and separation.

As an alternative, it is possible to target specific groups of proteins,such as proteins bearing post-translational modifications, in order tocompare differences in the post-translational modifications and otherdifferences occurring in proteins between two or more samples.

An example of such proteins is the glycoproteins. In recent years, thefunctional significance of carbohydrate on proteins has beenincreasingly realized. Carbohydrates are now known to be implicated inmany cellular and disease processes. It is possible to label theterminal carbohydrate groups of glycoproteins by first oxidizing theterminal sugars to aldehydes, followed by reaction with a hapten or afluor bearing a reactive group such as a hydrazide, as described byWilchek, M and Bayer, E. A., “Methods in Enzymology” vol. 138, 429-442(1987).

More specifically this would involve: Preparing an extract of two ormore protein samples, incubating each of said protein samples in thepresence of periodate for a short period to oxidise vicinal diols on theterminal sugars to aldehyde groups. Excess periodate would then bedestroyed by adding bisulphite, before labeling with a suitablefluorescent dye from a matched set. Alternatively, it is possible tospecifically label sialic acid residues by oxidation of their exocycliccarbons using a lower concentration of periodate, typically 1 mM andusing a temperature of 0° C. It is also possible to treat the proteinsample with galactose oxidase to generate an aldehyde group on terminalgalactose residues to form a C-6 aldehyde derivative, which can then bereacted with a suitable fluor. The reaction of the dye and thecarbohydrate may be quenched by the addition of suitable material beforemixing samples together and subjecting to a suitable separation method.

Fluorescent dyes that can be used for glycoprotein labeling includethose dyes having hydrazine derivatives such as hydrazides,semicarbazides and carbohydrazides or amine derivatives as reactivegroups. In order to maintain the overall charge on the glycoprotein,suitable dyes are those that bear an overall neutral charge. An overallneutral charge can be obtained by the addition of suitably chargedlinkers to the dye molecule to produce the desired overall charge.Suitable dyes would include neutral cyanine derivatives, BODIPY®fluorescent dye derivatives or other fluorescent derivatives availableas matched dye sets as described herein and possessing an overallneutral charge.

The method of the present invention is also applicable tophosphoproteins which may be specifically labeled with fluors.Phosphoproteins perform important biological roles, including cellsignaling, and are involved in a number of regulatory mechanisms. Thephosphate groups on proteins may be specifically labeled as, forexample, by the procedure described by Giese and Wang, U.S. Pat. No.5,512,486, incorporated herein by reference, using fluorescent or haptenimidazole derivatives. The labeling reagents bearing the imidazolegroups are added to the protein samples in the presence of a suitablecarbodiimide to effect labeling.

More specifically the method involves preparing an extract of two ormore protein samples, incubating each of said protein samples with asuitable water-soluble carbodiimide, plus a fluorescent imidazolederivative from a matched dye set. Although the carbodiimide willactivate side chain carboxyls as well as phosphates, the bond withcarboxyl groups is unstable when the pH is raised to around 8. Excessreactive material can be removed by the addition of a suitable knownquenching material. The samples are then mixed and separated by a knownseparation procedure.

In order to maintain the overall charge on the phosphoprotein, suitablefluorescent imidazole derivatives would bear an overall negative charge.This overall negative charge could be obtained by the addition of asuitably charged linker to the dye molecule. Examples of dyes that canbe used include, cyanine dyes bearing an overall negative charge,squarate dye derivatives bearing an overall negative charge or otherfluorescent derivatives bearing an overall negative charge and availableas matched dye sets. Preferred carbodiimide molecules are water solublemolecules such as (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride) (EDC) and1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide) (EAC).

The first group of dyes evaluated were the fluorescent cyanine dyesdescribed in Mujumdar, R. B. et al., “Cyanine dye labeling reagentscontaining isothiocyanate groups”, Cytometry 10:11-19 (1989) andWaggoner et al., U.S. Pat. No. 5,268,486 entitled “Method for labelingand detecting materials employing arylsulfonate cyanine dyes” issued in1993 and incorporated herein by reference. The cyanine dyes have thefollowing general structure.

where X and Y can be O, S or (CH₃)₂—C, m is an integer from 1 to 3 andat least one of R₁, R₂, R₃, R₄, R₅, R₆ or R₇ is a reactive group whichreacts with amino, hydroxy or sulfhydryl nucleophiles. The dotted linesrepresent the carbon atoms necessary for the formation of one ring tothree fused rings having 5 to 6 atoms in each ring. R₃, R₄, R₆ and R₇are attached to the rings. The reactive moiety can be any known reactivegroup. Reactive groups that may be attached directly or indirectly tothe chromophore to form R₁, R₂, R₃, R₄, R₅, R₆ or R₇ groups may includereactive moieties such as groups containing isothiocyanate, isocyanate,monochlorotriazine,, dichlorotriazine, mono- or di-halogen substitutedpyridine, mono- or di-halogen substituted diazine, maleimide,phosphoramidite, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide,glyoxal, ketone, amino and aldehyde.

Because of their intrinsic positive charge, the cyanine dyes describedin the Waggoner et al. patent are the fluorophors of choice when apositive charge is desired for labeling primary amines. The cyaninesattach to the protein via the activated ester of hexanoic acid. Whilethe coupling destroys the charge of the lysine side chain, the intrinsiccharge in the dye compensates. It in effect moves the charge away fromthe protein molecule but maintains the same overall charge within thesample to be electrophoresed. In the cyanine dye molecule, twofunctionalized indole rings are connected via a polyene linker. Thespectral characteristics of cyanine dyes can be easily modulated bysimply changing the length of the linker between the indole rings of thedye. A longer or shorter linker length will result in fluorescence atdifferent wavelengths and thus, different colors. However, changing thelength of the linker changes the molecular mass of the dye. Sinceelectrophoresis depends also on the mass of the proteins, the effect ofthe dye on a protein's mass can also be of concern. Because the proteinsare labeled before electrophoresing, the mass of the dye attached to theprotein must not significantly alter the relative differences in themolecular weights of the various proteins in the extracts. Molecularweight is not critical, however, because only a relatively small numberof sites on the protein are labeled. As indicated above, preferably lessthan 1%, up to about 2% of the possible attachment sites on the proteinsare labeled. If more are labeled, maintaining generally equal molecularweights for the dyes within the set of matched dyes becomes a greaterconcern.

The difference in molecular weight caused by changing the linker lengthin the fluorescent cyanine dyes can be compensated for by modulating thesize of an aliphatic chain R₁ or R₂, attached to one of the dye's indolerings. One of R₁ or R₂ must be a reactive group. These designconstraints led to the modification of the cyanines and the developmentof a dye of the general formula

wherein X and Y equal S, O, or CH₃—C—CH₃, m is an integer from 1 to 3and either R₁ or R₂ is a reactive group capable of covalently binding tothe protein, such as the reactive groups described above for theunmodified cyanine dyes. The dotted lines represent 1, 2 or 3 fusedrings having 5 or 6 carbon atoms in each ring. Each side should balancethe other side.

An example of a matched pair of dyes developed according to the generalformula follows:

(Propyl Cy-3-NHS) which fluoresces red and,

(Methyl Cy-5-NHS) which fluoresces far red in the spectrum wherein R isa reactive group. As stated above, O or S or a combination thereof canbe placed in the X and Y positions in place of (CH₃)₂C—.

The cyanine dyes are one choice for the matched set of dyes of thepresent invention. Other dye compounds may be used in place of thecyanines, such as dipyrromethene boron difluoride dyes, the derivatizedThe cyanine dyes are one choice for the matched set of dyes of thepresent invention. Other dye compounds may be used in place of thecyanines, such as dipyrromethene boron difluoride dyes, the derivatized4,4-difluoro-4-bora-3a,4a,-diaza-S-indacene dyes, described in U.S. Pat.No. 4,774,339 to Haugland et al. and incorporated herein by reference,which are sold by Molecular Probes, Inc. under the trademark BODIPY®.The BODIPY® fluorescent dyes, which have no net charge, are covalentlylinked to lysine side chains using an activated n-hydroxysuccinimidylester which forms an amide bond. The result is the loss of the lysinepositive charge. Therefore, a positively charged linker group is used inthe matched dyes of the invention to replace the lost primary amine withthe linker's tertiary amine. The procedures for making BODIPY®fluorescent dyes are described in U.S. Pat. No. 4,774,339. Addition, ofthe positively charged linker is by techniques well known to thoseskilled in the art. A linker can be designed with three functionalgroups; (1) to react with the BODIPY®-NHS ester, (2) to carry thedesired charge, and (3) to be activated so that the BODIPY®-linkerconstruct will react with specific amino acid residues of the proteinsin the extract.

The major considerations for the matched set of dyes are the maintenanceof charge and distinct and different spectral characteristics. Anyneutral dyes with a positive linker or any positively charged dyes,preferably each having a +1 charge, that otherwise satisfy therequirements described herein can serve as the dyes in the matched setof dyes of the present invention. Roughly equal molecular weight in thesamples of labeled protein is desirable, but as explained above, notcritical. The intrinsic positive charge of cyanine dyes isadvantageously used in the preferred embodiment to replace the positivecharge of lysine. The pK_(a) of cyanines and lysine are ratherdifferent; however, conditions were selected for dye:protein ratio to beless than one. This low level of labeling ensures that there will benegligible changes in the protein's migration on two-dimensionalelectrophoresis gels. Dyes may be used which match the pK_(a) of lysinemore closely. Alternately, dyes that modify other amino acid residuesmay be used, provided the amino acid's ionic characteristics arepreserved by the modification. Instead of a lysine, the attachment siteon the protein may be a sulfhydryl or carboxylic group. When asulfhydryl group is the attachment site on the protein, thecorresponding attachment site on the dye is an iodoalkyl or maleimidegroup. When a carboxylic acid group is the attachment site on theprotein, the corresponding attachment site on the dye is a chloroketoneor a carbodiimide.

It is anticipated that the method of the present invention also can beused to detect the presence of different nucleic acids in differentsamples. The charge of nucleic acids is very negative. The addition ofthe dye does not therefore alter the overall charge in nucleic acids sothe choice of the matched set of dyes does not have to compensate forcharge loss when nucleic acid analysis is contemplated. To facilitateattachment of the dye, nucleic acids can be modified to have a freeamino acid coming from the nucleic acid nucleus by techniques known tothose skilled in the art. A lysine would be suitable in this instancealso.

EXAMPLE 1

Synthesis of the Dyes (Methyl Cy-5 and Propyl Cy-3)

1. Synthesis of Indole Derivatives (Common to Both Dyes)

4.8 g (30 mmoles) of 2,3,3-trimethyl-(3H)-indole and 35 mmoles of thedesired bromoalkyl reagent (6-bromohexanoic acid or 1-bromopropane) in40 ml of 1,2-dichlorobenzene were heated to 110° C. under nitrogen gasand stirred overnight with refluxing. The product (acid indole, methylindole, or propyl indole) precipitated as an orangish gum. Thesupernatant was decanted and the gum was washed several times with ethylether. This intermediate was used as is.

2. Cy-3 Intermediate

1.5 g (7.5 mmoles) of propyl indole was added to 1.6 g (7.6 mmoles) ofN-N′ diphenyl formamidine in 20 ml glacial acetic acid and was refluxedfor 4 hrs. The solvent was removed under vacuum leaving a deep orangesyrup. This intermediate was used as is.

2a. Cy-5 Intermediate

The synthesis of the Cy-5 intermediate is the same as the synthesis ofthe Cy-3 intermediate in step 2 of the dye synthesis except that2-methylene-1,3,3-trimethylindoline was used instead of propyl indoleand the linker was malonaldehyde dianil. The gummy, bluish intermediatewas washed twice with ethyl ether.

3. Cy-3

2.5 ml of triethylamine and 1.8 ml of anhydrous Ac₂O were added to theintermediate from step 2., and the mixture was boiled for 5 minutes.1.70 g (5.0 mmoles) of acid indole was added and the mixture wasrefluxed for two hours. The solvent was removed under vacuum and theproducts were dissolved in 10 ml of EtOH.

3a. Cy-5

The preparation of Cy-5 is the same as that of Cy-3 except that theintermediate from step 2a. was used instead of the intermediate fromstep 2.

4. Purification of the Products from Steps 3. and 3a.

Methyl Cy-5 and propyl Cy-3 were separated from contaminating sideproducts by running flash chromatography with a silica gel solid phaseand 40% MeOH in dichloromethane as the mobile phase.

5. Activation of Carboxyl Groups

The carboxylic acid moiety of each dye was converted into anN-hydroxysuccinimidyl ester by dissolving a quantity of purifiedmaterial in 5 ml of dry dimethylformamidine (DMF). 1.5 equivalents ofN-N′ disuccinimidyl carbonate (DSC) was added with 0.1 ml drypyridine/100 mg dye. The reaction was refluxed at 60° C. for 90 minutesunder nitrogen.

EXAMPLE 2

Protein Labeling

1. Bacterial Culture

Initial experiments were performed on E. coli that expressed thechimeric GAL4VP16 protein under the control of the lac promoter asdescribed in Chasman, D. I. et al., “Activation of yeast polymerase IItranscription by Herpesvirus VP16 and GAL4 derivatives in vitro”,Molecular Cell Biology 9:4746-4749 (1989). Two cultures of bacteria weregrown to an OD600 of 0.7 at 37° C. in 125 ml of standard LB mediumcontaining 50 μg/ml ampicillin. Isophenylthiogalactopyranoside (IPTG), anon-hydrolyzable analog of lactose, was added to one culture at a finalconcentration of 1 mM. Both cultures were incubated for an additional2.5 hours.

2. Protein Isolation for Two-dimensional Gel Electrophoresis

Isolation of protein was as follows. The bacteria was isolated bycentrifugation. Each bacterial pellet was washed with sonication buffercontaining 5 mM Hepes KOH pH 8.4, 5 mM Mg(OA_(c))2. The pellet wasresuspended in sonication buffer containing 50 μg/ml RNase to a finalvolume of 100 μl. This was then sonicated in ice until the solution wasclear, usually several minutes. DNase was added to 50 μg/ml and thesample was incubated for 30 min at 0° C. Solid urea and CHAPS were addedto a final concentration of 8 M and 5% respectively. The sample wastaken off the ice and 1 volume of lysis buffer added. The sample waseither labeled immediately or stored at −80° C.

3. Protein Labeling

Propyl Cy-3-NHS was added to the first sample and Methyl Cy-5-NHS wasadded to the second sample of cell extract at a concentration of 2 mmoleof dye/50 μg of protein. The dye stock solution was typically 2 mM indimethyl formamide. The reaction was incubated at 0° C. for 30 minutes.Incubation times may vary from about 15 to about 30 minutes, dependingon the temperature and the type of cells being studied. Incubation canbe for 15 minutes when the temperature is about 25° C. The temperatureshould not be above that which will cause the proteins to be degraded.The labeled sample was immediately subjected to isoelectric focusing orstored at −80° C.

4. Protein Isolation and Labeling for SDS-gel Electrophoresis

Bacteria were grown and isolated by sonication as in step 2, of theprotein labeling procedure, except RNase or DNase was not added. Thecell extract was directly labeled as in step 3 of the protein labelingprocedure. SDS, glycerol, Tris HCl pH 6.8, and bromophenol blue wereadded to bring the final concentrations to 1%, 10%, 64 mM, and 5 μg/ml,respectively. The sample was then placed in a boiling water bath for 2minutes and then subjected to electrophoresis.

5. Determination of Dye to Protein Ratio

In order to prevent solubility problems with labeled proteins,conditions were chosen to only label 1-2% of the lysines in the cellextract. This is based on the assumption that 7% of an average protein'samino acids are lysine. The first step in determining the dye to proteinratio was the removal of free dye by adsorption to SM-2 beads (Bio-Rad).The protein concentration was determined by OD260/280. The dye contentwas determined by OD548 and OD650 for Propyl Cy-3 and Methyl Cy-5,respectively (=100,000 for both dyes).

EXAMPLE 3

Gel Electrophoresis

1. Two-dimensional Electrophoresis

High resolution two-dimensional gel electrophoresis was carried out bywell known techniques.

2. SDS Polyacrylamide Gel Electrophoresis

SDS polyacrylamide gel electrophoresis was carried out by knowntechniques.

EXAMPLE 4

Fluorescence Gel Imaging

At the end of electrophoresis, the gels were soaked in a solution of 25%methanol and 7% acetic acid. The fluorescently labeled proteins in thegel were imaged in the following manner. Gels were placed on a surfaceof anodized aluminum and irradiated at an incident angle of 60° with a300 W halogen lamp housed in a slide projector. The light exiting theprojector was passed through 1′ diameter bandpass filters (ChromaTechnologies, Brattleboro Vt.), 545±10 nm and 635±15 nm for Cy-3 andCy-5, respectively. The images were collected on a cooled, CCD camera(Photomctrics Inc., Tucson Ariz.) fitted with a 50 mm lens (Nikon) and adouble bandpass emission filter (Chroma Technologies, Brattleboro Vt.),587.5±17.5 nm and 695±30 nm for Cy-3 and Cy-5, respectively. The CCDcamera was controlled by a Macintosh II si computer running Photometricscamera controller software. Image integration time ranged from tenths ofseconds to several minutes. The excitation filters were housed in afilter wheel attached to the projector. Two successive images wererecorded with irradiation from the two filters without moving the gel.

EXAMPLE 5

Image Processing

The image files were transferred to a Personal Iris 4D/35 (SiliconGraphics Inc., Mountain View Calif.). The image files were thenprocessed using the DeltaVision™ software (Applied Precision, MercerIsland Wash.). The two schemes were used to determine the differencesbetween the differently labeled samples on the gel:

1. Subtraction

Each image can be considered as a grid-like array of pixel intensities.These arrays of values can be manipulated by a number of arithmeticoperations. Here one image was subtracted from the other. Because thetwo samples loaded onto the gel were not perfectly balanced for overallfluorescence, one image was multiplied by a balancing constant. Thisfactor was determined arbitrarily so that the number of differencesbetween the samples were kept small.

2. Ratio Imaging

Here one image was divided by the other. Before this operation wasperformed the images were first normalized to a common intensity range.This was done by setting the minimum and maximum pixel values of eachimage to zero and an arbitrarily large value, 4095, the maximum possibleoutput value of the CCD camera employed. Intermediate pixel values werescaled linearly between these values. One image was then divided by theother. A balancing factor was also used here to keep the mean quotientat one. Regions of difference were those with a quotient greater thanone.

EXAMPLE 6

1. Difference SDS Gel Electrophoresis of Induced GAL4VP16 Expression inBacteria

FIG. 2 shows images of Propyl Cy-3 and Methyl Cy-5 labeled proteins runon a single SDS polyacrylamide gel.

Lanes 1-3 show Cy-3 labeled protein. The samples loaded in there laneswere:

Lane 1. Propyl Cy-3 labeled IPTG-induced bacterial extract.

Lane 2. Propyl Cy-3 labeled IPTG-induced bacterial extract plus MethylCy-5 labeled uninduced extract.

Lane 3. Propyl Cy-3 labeled purified GAL4VP16 protein.

Lanes 4-6 show Cy-5 labeled protein. The samples loaded in there laneswere:

Lane 4. Propyl Cy-3 labeled IPTG-induced bacterial extract.

Lane 5. Propyl Cy-3 labeled IPTG-induced bacterial extract plus MethylCy-5 labeled uninduced extract.

Lane 6 Propyl Cy-3 labeled purified GAL4VP16 protein.

Only Lane 5 showed Cy-5 fluorescence.

Lanes 7 and 8 show the subtracted product of Lane 2-Lane 5 and Lane3-Lane 6, respectively. The arrows point to the position of GAL4VP16 asconfirmed by the position of the purified GAL4VP16 band in lane 8. Theidentity of the upper bands is not known. However, there are severalproteins that are known to be induced by IPTG, includingβ-galactosidase.

Lanes 9-11 show Cy-5 labeled-protein. The samples loaded in these laneswere:.

Lane 9. Methyl Cy-5 labeled IPTG-induced bacterial extract.

Lane 10. Methyl Cy-5 labeled IPTG-induced bacterial extract plus PropylCy-3 labeled uninduced extract.

Lane 11. Methyl Cy-5 labeled purified GAL4VP16 protein.

Lanes 12-15 show Cy-5 labeled protein. The samples loaded in there laneswere:

Lane 12. Methyl Cy-5 labeled IPTG-induced bacterial extract.

Lane 13. Methyl Cy-5 labeled IPTG-induced bacterial extract plus PropylCy-3 labeled uninduced extract.

Lane 14. Methyl Cy-5 labeled purified GAL4VP16 protein.

Only Lanes 12-15 all showed some Cy-3 fluorescence. This is due toslight crossover between the bandpass filters. This causes Cy-5 labeledmaterial to appear when excited by Cy-3 light. The converse is not seen.Cy-3 material is not visualized by Cy-5 excitation light. There are twoways to eliminate the crossover effects: design better bandpass filtersor computationally remove the Cy-5 contribution to the Cy-3 image byknowing the crossover constant. Lanes 15 and 16 show the subtractedproduct of Lane 10-Lane 13 and Lane 11-Lane 14, respectively. The arrowspoint to the position of GAL4VP16 as confirmed by the position of thepurified GAL4VP16 band in Lane 16. The identity of the upper bands isnot known. However, there are several proteins that are known to beinduced by IPTG, including β-galactosidase.

2. Difference Two-dimensional Gel Electrophoresis of Induced GAL4VP16expression in Bacteria

FIG. 3 shows images of a portion, of a two-dimension gel loaded withPropyl Cy-3 labeled IPTG-induced bacterial extract plus Methyl Cy-5labeled uninduced extract.

Panel A. Images taken with Cy-3 excitation light showing theIPTG-induced proteins.

Panel B. Images taken with Cy-5 excitation light showing the uninducedproteins.

Panel C. Ratio of the Cy-3 image divided by the Cy-5 image.

Panel D. Overlay of the image in Panel C, colored red, and placed on topof the image from Panel B, colored blue.

3. Difference Two-dimensional Gel Electrophoresis of Bacteria Extractwith Exogenously Added Protein

FIG. 4 shows images of a portion of a two-dimension gel loaded withPropyl Cy-3 labeled bacterial extract that had exogenously addedcarbonic anhydrase plus Methyl Cy-5 labeled extract without the addedcarbonic anhydrase.

Panel A. Image taken with Cy-3 excitation light showing the bacterialproteins plus carbonic anhydrase.

Panel B. Images taken with Cy-5 excitation light showing the bacterialproteins alone.

Panel C. Ratio of the Cy-3 image divided by the Cy-5 image.

Panel D. Overlay of the image in Panel C, colored red, and placed on topof the image from Panel B, colored blue.

The process of the present invention provides a simple and inexpensiveway to analyze the differences in protein content of different cells ordifferent samples from other sources. The process eliminates problemswhich can occur using two separate gels which must be separatelyelectrophoresed. The matched dyes used to label the different proteinsallow simultaneous electrophoresis of two or more different samples in asingle gel. While the invention has been described with reference to twosamples of proteins and a matched pair of dyes, those skilled in the artwill appreciate that more than two samples may be simultaneously testedusing an equal number of matched dyes. As long as the spectralcharacteristics of the dyes can be manipulated to provide fluorescenceat a number of different wavelengths resulting in visually distinctimages and the pH and ionic characteristics of the dyes can be generallyequalized to compensate for changes made to the protein by virtue ofcovalent bonding to the dye, multiple dyes can be used.

Differential Analysis of Glycoproteins

Dye Synthesis

EXAMPLE 7

Synthesis of the cyanine dye intermediates was as previously described.The carboxylic acid moiety of each of the Cy3 and Cy5 intermediates werethen converted to hydrazide as detailed below.

To a stirred solution of Cy-3 (50 mg, 8.9×10⁻⁵ mol) dissolved inanhydrous acetonitrile (2 ml) under a nitrogen atmosphere was addeddiisopropylamine (0.03 ml, 9.7×10⁻⁵ mol) andO—(N-succinimidyl)-N,N,N′,N′-bis(tetramethyl)uronium tetrafluoroborate(TSTU) (30 mg, 9.7×10⁻⁵ mol) and the reaction stirred at ambienttemperature for 1 hour. Analysis of the material by thin layerchromatography (TLC) revealed that none of the starting materialremained so an additional equivalent of diisopropylamine (0.03 ml,9.7×10⁻⁵ mol) was added which was then subsequently followed bytertbutyl carbazate (30 mg, 1.78×10⁻⁴ mol). The reaction was stirredovernight at ambient temperature then the solvent removed in vacuo togive an intensely colored pink oil. The oil was purified using flashcolumn chromatography (silica:dichloromethane/methanol gradient)resulting in a pink solid (59 mg, 97%). The product was clean andcorrect by ¹H NMR and UV/VIS spectrometry (λ_(max)=552 nm).

Chloroform (1 ml) was added to a portion of the pink solid (5 mg) and aturbid solution resulted which was treated with trifluoroacetic acid (4drops). After 1 hour incubation, TLC revealed that all the product hadbeen consumed and a more polar product had been formed so the solventswere removed in vacuo and the resultant semi-solid was triturated withdiethyl ether then dried in vacuo. The product appeared clean andcorrect by ¹H NMR and UV/VIS spectrometry (λ_(max)=552 nm).

EXAMPLE 8

The preparation of Cy5 hydrazide is the same as that of Cy3 except thatthe starting material is Cy5.

EXAMPLE 9

Protein Labeling

1) Cell Culture

Initial experiments were performed on cultures of HBL 100 human breast,(see, In Vitro Cell & Dev. Biol., vol. 26, 933 (1990)), and BT474 humanbreast ductal carcinoma, (J. Natl. Cancer Inst. (Bethesda), vol. 61, 967(1978)), epithelial cell lines. HBL100 cells were grown to confluence asa monolayer in McCoy's 5A media supplemented with 10% fetal bovine serumand 2 mM glutamine at 37° C. in 5% CO₂ in air atmosphere. BT474 cellswere grown to confluence as a monolayer in RPMI 1460 media supplementedwith 10% fetal bovine serum, 2 mM glutamine, 0.02 mg/ml bovine insulin,0.45% glucose and 1 mM sodium pyruvate at 37° C. in 5% CO₂ in airatmosphere.

2) Protein Isolation from Cells

Flasks of cell monolayers were washed twice with PBS to remove media andthe cells harvested by incubation in trypsin. The cell suspension wascentrifuged at 2000 rpm for 5 minutes to pellet the cells. Thesupernatant was discarded and the cell pellet was washed with Tris toremove excess salt. The cell pellets were stored at −70° C. Protein wasextracted from the cells by sonication. The cell pellets (˜2×10⁶cells/ml) were resuspended in lysis buffer containing 2 M urea, 100 mMacetate buffer pH 5.5, 1% (v/v) NP-40 and 0.1% (w/v) SDS and sonicatedon ice 4 times each for 20 seconds. The cell lysates were centrifuged at4° C. at 13,000 rpm in a microfuge for 5 minutes to remove cell debrisand the supernatant retained. Cell supernatants were stored at −20° C.

3) Protein Concentration Determination for Estimation of Dye:ProteinRatio

In order to prevent solubility problems with labeled proteins and toenable use of consistent dye:protein ratios, the concentration ofextracted protein was determined by the BioRad Dc protein assay (BioRadLaboratories).

4) Labeling of Carbohydrate on Glycoproteins Using Hydrazide Dyes

a) Labeling of model glycoproteins for SDS-PAGE

i) Solutions of individual glycoproteins were prepared as 10 mg/mlstocks in water. 10 μg of each protein was taken for labeling anddiluted with acetate buffer pH 5.5 to a final buffer concentration of100 mM.

ii) Sugar residues were oxidized by addition of sodium metaperiodate inwater to give a final concentration of 10 mM and incubated for 20minutes at ambient temperature in the dark. Excess metaperiodate wasremoved by addition of sodium metabisulphite in 200 mM acetate buffer pH5.5 to give a final concentration of 5 mM and incubated for 5 minutes atambient temperature.

Cy3 hydrazide was added to the first cell sample and Cy5 hydrazide wasadded to the second cell extract at a concentration of ˜25 nmol/10 μgprotein. The dye stock solution was typically 10 mM indimethylformamide. The labeling reaction was incubated at ambienttemperature for 30 minutes. Incubation times may vary from about 10 to60 minutes and the incubation temperature may vary from 0° C. to ˜25° C.depending on the type of cells being studied. The temperature should notbe above that which will cause the proteins to be degraded and the timeshould not be longer than that which could cause non-specific labeling.

iii) Gel loading sample buffer was added to labeled samples to givefinal concentrations of 2% (w/v) SDS, 10% (v/v) glycerol, 62.5 mMTris-HCl pH 6.8, 5% (v/v) mercaptoethanol, 0.01% bromophenol blue. Thesample was placed in a boiling water bath for 4 minutes then subjectedto electrophoresis.

b) Labeling of simple glycoprotein mixtures for SDS-PAGE Simple mixturesof 12 or 14 different model proteins were prepared containing ˜10 μgeach of proteins from 10 mg/ml protein stock solutions to give a ladderof molecular weights. The protein solution was diluted 1:1 with 200 mMacetate buffer pH 5.5. The protein mixes were labeled according to theprocedure described in step 4a (ii) above. Samples were analyzed bySDS-PAGE after addition of sample buffer as described.

c) Labeling of glycoproteins in cell extracts for 2-dimensional gelelectrophoresis. Protein was extracted from cells as described in step 3of this example and used directly for labeling of carbohydrate groups onglycoproteins. ˜150 μg of extracted protein was labeled as described instep 4a (ii) above. Labeled samples (˜25-50 μg) were mixed with 2×IEFsample buffer (8M urea, 4% (w/v) CHAPS, 2% (v/v) Pharmalytes, 20 mg/mlDTT) and loaded directly onto isoelectric focusing strips (ImmobilizedpH gradients, Amersham Pharmacia Biotech).

5) Labeling of Lysine Residues in Proteins Using NHS Dyes

Proteins were labeled on lysine residues with amine reactive NHS dyes aspreviously described using 200 pmol of Cy2-NHS ester/50 μg protein in 5mM Tris buffer. Proteins from cell extracts were directly labeled withCy2 as previously described.

6) Gel Electrophoresis

1. Two dimensional electrophoresis: high resolution two-dimensionalelectrophoresis was carried out by well known techniques according toLaemmli [Nature, vol. 227, 680-685 (1970)].

2. SDS-polyacrylamide gel electrophoresis: SDS-polyacrylamide gelelectrophoresis was carried out by known techniques.

7) Fluorescence Gel Imaging

At the end of electrophoresis, the fluorescently labeled proteins in thegels were imaged using commercially available scanners with appropriateexcitation and emission wavelengths [Cy2 excitation 480/30 emission530/30; Cy3 excitation 540/25, emission 590/35; Cy5 excitation 620/30,emission 680/30].

EXAMPLE 10

Labeling of Model Glycoproteins

FIGS. 5a) and b), respectively, show images of Cy2-NHS and Cy3 hydrazidelabeled proteins run on SDS-PAGE.

Lanes 1 and 3 show proteins labeled at lysine residues with Cy2 NHSester visualized with Cy2 excitation and emission.

Lane 1. Cy2 labeled transferrin (molecular weight ˜76 kDa).

Lane 3. Cy2 labeled soybean trypsin inhibitor (molecular weight ˜20kDa).

Lanes 2 and 4 show the same proteins labeled a carbohydrate groups withCy3 hydrazide visualized with Cy3 excitation and emission.

Lane 2. Cy3 labeled transferrin.

Lane 4. Cy3 labeled soybean trypsin inhibitor.

Lanes 1 and 3 show that both transferrin and trypsin inhibitor arelabeled with the Cy2 lysine dye. Lane 4 shows labeling of transferrinwith Cy3 hydrazide. The trypsin inhibitor in lane 4 is not visible withCy3 as this protein is not glycosylated and therefore does not labelwith Cy3.

EXAMPLE 11

Differential SDS-PAGE Analysis of Simple Protein Mixes

FIGS. 6a) and b) show images of Cy3 (6 a) and Cy 5 (6 b) hydrazidelabeled protein mixes run on SDS-PAGE. Protein mix B was prepared bylabeling 10 mg of each of the following proteins—fetuin, albumin,carboxypeptidase Y, ribonuclease B, α-1 acid glycoprotein, trypsininhibitor, lactoglobulin, cytochrome C, α-lactalbumin, lysozyme,myoglobin and actin. Protein mix A contains identical proteins to mix Bplus transferrin and carbonic anhydrase.

Lanes 1 and 3 of FIG. 6(a) show Cy3 labeled protein mixes A and B.

Lanes 2 and 4 of FIG. 6(b) show Cy5 labeled protein mixes A and B.

Lane 5 shows Cy3 labeled protein mix A plus Cy5 labeled protein mix B.

Lane 6 shows Cy5 labeled protein mix A plus Cy3 labeled mix B.

Lanes 5 and 6 with the protein mixes run in the same lane showdifferential detection of the two additional proteins in mix A—in lane 5visible on the Cy3 image and in lane 6 visible on the Cy5 image.

EXAMPLE 12

Differential 2DE Analysis of Mammalian Cell Extracts

FIGS. 7(a) and (b) shows images of a section of a 2DE gel loaded withCy3 (7 a) hydrazide labeled HBL100 cell extract and Cy5 (7 b) hydrazidelabeled BT474 cell extract. Differences in the glycoprotein content ofthe cell lines are apparent from the different pattern of spots obtainedwith the two cell extracts. Some of the qualitative and quantitativedifferences have been highlighted with arrows.

EXAMPLE 13

Differential 2DE Analysis of Mammalian Cell Extract with ExogenouslyAdded Protein

Cell extracts were prepared as described in steps 1-4 of Example 9above. To simulate differences in complex cell lysates the cell extractswere labeled with Cy3 and Cy5 hydrazide and Cy5 labeled knownglycoproteins were added into Cy5 labeled sample. The Cy3 and Cy5labeled samples were than mixed in equal volumes before 2DE. FIGS. 8a)and b) show images of a 2DE gel loaded with Cy3 (8 a) labeled cellextract (without exogenously added protein) and Cy5 (8 b) labeled cellextract with exogenously added protein. The added proteins were asfollows:

Protein Molecular weight (kDa) pI transferrin ˜76 ˜6.3 fetuin ˜70 ˜5carboxypeptidase-Y ˜58 6.3 (predicted)* α-1 acid glycoprotein ˜45 ˜4.2albumin ˜45 ˜5.5 carbonic anhydrase ˜30 7.9 (predicted) *From Prositedatabase

The section of gel highlighted shows proteins ranging in molecularweight from ˜15-80 kDa and pH range 5-8. This portion thus excludes theproteins fetuin, α-1 acid glycoprotein and albumin from being detectedin the Cy5 image.

The Cy3 and Cy5 images show good reproducibility in the spots detectedusing the two dyes to label the same BT474 cell sample and thedifferences can be attributed to the exogenously added proteins.Transferrin and carbonic anhydrase are highlighted on the Cy5 image butare not visible on the Cy3 image. The presence of carboxypeptidase-Y inthe Cy5 sample has not been clearly demonstrated but this may be due toa number of causes such as overlap with endogenous cell proteinspreventing it from being resolved or low level of carbohydrate on theprotein.

Differential Protein Analysis by Column Chromatography EXAMPLE 14

Protein Labeling

Proteins were labeled by means of a minimal labeling approach designedto add a single label onto each protein. This procedure results in onlya small proportion of each protein being labeled. Protein levelsindicated in the text below are total protein amount and do not takeinto account the proportion of protein actually labeled. Dyes containinga single net positive charge were reacted with primary amine groups onthe protein to avoid change in overall charge status of the protein.

The N-hydroxysuccinimidyl esters of Cy3 and Cy5 derivatives matched formolecular weight (Amersham Pharmacia Biotech) containing a singlepositive charge were added to buffer (20 mM Tris/HCl pH7.6) containingprotein or peptide at a ratio of 800 picomole dye per 200 microgram ofprotein in a total volume of 46 μl. The solution was incubated on ice inthe dark for 30 minutes. The reaction was then stopped by the additionof 4 μl of 10 mM lysine solution and incubation on ice for a further 10minutes.

Purified proteins were either labeled separately or mixed and thenlabeled. Anti-mouse immunoglobulin G (IgG) was obtained from thesupplier (Amersham Pharmacia Biotech) ready labeled with either Cy3 orCy5.

Instrumentation

An FPLC system (Amersham Pharmacia Biotech) consisting of pumps, valvesand controller was used to pump samples through chromatography columns.The eluant from each column was fed into an 8 μl quartz flow-throughcell (Hellma) positioned in an F4500 fluorimeter (Hitachi). The opticswere not, however, optimized since the flowthrough cell had a verticalwindow and the light beam had a horizontal alignment, limiting thepotential sensitivity of this particular system. Software designed tointerrogate 2 excitation and emission wavelengths was used to allowcontinuous monitoring of the presence of Cy3 and Cy5 labeled proteins.For detection of Cy3, wavelength settings used were 530 nm (excitation)and 570 nm (emission). For detection of Cy5, settings were 630 nm(excitation) and 670 nm (emission). Slit widths for excitation andemission were 10 nm.

Chromatography

Cy3 and Cy5 labeled proteins or peptides were detected simultaneouslyand continuously on elution from chromatography columns. Mixtures ofproteins or peptides were profiled on a variety of column types.Differences between two protein samples could also be examined bylabeling the samples with different fluors and then mixing followed bychromatography and simultaneous detection of the fluorescence of the twofluors.

EXAMPLE 15

Ion Exchange Column

MonoQ HR5/5 anion exchange column (50×5 mm internal diameter) (AmershamPharmacia Biotech) was equilibrated with starting buffer (20 mM Tris/HClpH 7.6). Protein samples made up in starting buffer were applied to thecolumn using a 100 μl loop. Proteins were eluted from the column using alinear gradient of the same buffer containing 350 mM NaCl over a 25minute period at a flow rate of 0.5 ml per minute. Cy3 and Cy5 labeledproteins were detected simultaneously and continuously on elution fromthe ion exchange column.

FIG. 9 shows the separation of Cy3 labeled bovine serum albumin (BSA)and Cy5 labelled transferrin.

FIG. 10 shows the separation of the mixture of Cy5 labeled myoglobin,transferrin and bovine serum albumin.

Differential analysis by ion exchange chromatography and fluorescencedetection is shown in FIG. 14. One sample containing Cy3 labeledtransferrin has been mixed with another sample containing Cy5 labeledtransferrin and Cy5 labeled bovine serum albumin (BSA). The absence ofBSA from the second sample is clear.

EXAMPLE 16

Reverse Phase Column

A ProRPC column (silica based C1/C8 mix)(100×5 mm internal diameter) wasequilibrated with a mix of eluant A (70%) and eluant B (30%). Eluant Acontained 0.1% trifluoroacetic acid, 0.1% triethylamine in water (95%)acetonitrile (5%). Eluant B contained 0.1% trifluoroacetic acid, 0.1%triethylamine in water (25%) acetonitrile (75%). Protein samples made upin eluant A were applied to the column using a 100 μl loop and elutedusing a linear gradient from 70% eluant A/30% eluant B to 20% eluantA/80% eluant B over a 25 minute period at a flow rate of 0.5 ml perminute. Cy3 and Cy5 labeled proteins were detected simultaneously andcontinuously on elution from the reverse phase column.

FIG. 11 shows the reverse phase separation of the mixture of Cy5 labeledribonuclease A, cytochrome C, holo-transferrin and apomyoglobin.

EXAMPLE 17

Size Exclusion Column

A Superose 6 HR10/30 column (300×10 mm internal diameter) (AmershamPharmacia Biotech) was equilibrated with 10 mM phosphate buffered saline(PBS) pH 7.4. Protein samples made up in the same buffer were applied tothe column using a 100 μl loop and eluted at a flow rate of 0.5 ml perminute. Cy3 and Cy5 labeled proteins were detected simultaneously andcontinuously on elution from the size exclusion column.

FIG. 12 shows the separation of the mixture of Cy3 labeledthyroglobulin, apoferritin, IgG and β-lactoglobulin by size exclusionchromatography.

FIG. 13 shows differential analysis by size exclusion chromatography.The Cy3 labeled sample contains four proteins (thryoglobulin,apoferritin, IgG and β-lactoglobulin) and the Cy5 labeled sample showsjust three of the four proteins (minus IgG). The absence of the fourthprotein is clear.

EXAMPLE 18

Differential Protein Analysis by Affinity Purification

Cell Growth, Lysis and Affinity Purification

Lysates of E coli bacteria either expressing GST or not expressing GSTwere used as models of complex protein samples. E coli strain JM109cells were transformed either with plasmid pGEX-5X which encodes GST orwith control plasmid pTrc99 which does not encode GST (plasmids fromAmersham Pharmacia Biotech) and plated out onto agar plates. Liquidcultures (10 ml) were then grown overnight at 37° C. in LB brothcontaining 100 μg/ml ampicillin. Fresh media (typically 150 ml) was theninoculated with 0.5% starting culture and grown to an A₆₀₀ of 1.0. Thecells were then induced by addition of IPTG to a final concentration of0.5 mM and incubating for a further 2 to 3 hours. Cells were harvestedby centrifugation at 2800 g, taken up in 7.5 ml PBS and lysed bysonication (MSE 150 Soniprep) on ice (30 seconds, rest for 90 seconds,repeated 3 times). Cell debris was removed by centrifugation at 2800 gand after transfer of the supernatant to a fresh tube the centrifugationwas repeated. The supernatant (100 μl aliquot normalized with respect toconcentration determined by A₂₈₀ reading) was then labeled with 1.5 nmolCy dye on ice in the dark for 60 minutes and quenched with lysine asabove.

At this point, the lysates were either treated separately or they weremixed, for example a sample of pGEX cell lysate labeled with Cy3 beingmixed with an equal volume of pTrc cell lysate labeled with Cy5. GST wasthen affinity purified from the cell extract using Glutathione Sepharose4B (cat. no. 17-0756-01, Amersham Pharmacia Biotech) using the batchmethod protocol provided by the supplier for fusion protein screening.

Samples were read in the Hitachi fluorimeter exciting Cy3 at 530 nm(emission peak detected at 565 nm) and exciting Cy5 at 630 nm (emissionpeak detected at 661 nm) using 5 nm slits for excitation and emission.Material which did not bind to the affinity matrix was combined withwashings for fluorescence determination. Material bound to the affinitymatrix was eluted using excess unlabeled GST. Prior to reading, a 15 μlaliquot of each sample was diluted into a total volume of 100 μl. Washtrough samples were diluted a further 10 fold prior to reading becauseof their relatively high values. Subsequently, readings were correctedto take into account the relative volumes and dilutions of bound andwash through fractions.

Affinity Purification

Differential analysis by affinity purification was used to identify aspecific protein (glutathione-S-transferase, GST) in a cell lysate.Initial experiments demonstrated that in bacterial cell lysates labeledwith either Cy 3 or Cy 5 and analysed separately, the presence of GSTcould be determined. Samples from induced bacteria containing the pGEXplasmid which encodes for GST clearly contained GST protein as indicatedby fluorescent material specifically eluted from the affinityglutathione matrix with free glutathione (Table 1). This material wasconfirmed to be GST by imaging the fluorescence of an SDS-PAGE gelcontaining Cy5-labeled purified CST (Sigma) and affinity purified GST asdescribed above. Samples from induced bacteria containing the pTrcplasmid which does not encode for GST but is otherwise very similar, didnot appear to contain GST protein. This conclusion is indicated by a lowlevel of fluorescent material eluted from the affinity matrix and theabsence of a fluorescent band of correct molecular weight on an SDS-PAGEgel.

TABLE 1 Affinity purification of GST from cell lysates labeled eitherwith Cy3 or Cy5, lysates processed separately for affinity purification.Experiment 1 Experiment 2 Relative Cy3 Relative Cy5 fluorescencefluorescence Sample (F₅₆₅)* (F₆₆₁)* pGEX cell lysate fraction bound to62.1 99.8 affinity matrix pTrc cell lysate fraction bound to 2.6 5.3affinity matrix pGEX cell lysate washed through 13118 13487 affinitymatrix pTrc cell lysate washed through 13232 16320 affinity matrix*Readings corrected for differences in dilution and volume.

Subsequent experiments showed that when two separate lysates werelabeled with different dyes and mixed prior to affinity purification,differences in the GST content of the two samples could be determined bymeans of their relative fluorescence. For example, Cy3-labeled inducedpGEX cell lysate was mixed with Cy5-labeled induced pTrc cell lysate andaffinity purified. The proportion of material bound contained in the GSTexpressing cells was more than 10 times higher than that in cells notexpressing GST (Table 2). In the next sample, Cy3-labeled induced pGEXcell lysate was mixed with Cy5-labeled non-induced pGEX cell lysate andaffinity purified. The proportion of material bound contained in the GSTexpressing cells was again more than 10 times higher than that in cellsnot expressing GST (Table 2).

TABLE 2 Affinity purification of GST from cell lysates labeled eitherwith Cy3 or Cy5, lysates mixed prior to affinity purification.Proportion Proportion bound bound Sample (%) (%) Material fromCy3-labeled induced pGEX 3.3 0.28 cell lysate mixed with Cy5-labeledinduced pTrc cell lysate bound to affinity matrix Material fromCy3-labeled induced pGEX 3.8 0.32 cell lysate mixed with Cy5-labelednon- induced pGEX cell lysate bound to affinity matrix

What we claim is:
 1. A method of comparing protein compositions between at least two different cell samples comprising: (a) preparing an extract of proteins from each of said at least two cell samples; (b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal; (c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins; (d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins; (e) separating the dye-labeled proteins of interest within said mixture; and (f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection.
 2. The method of claim 1 wherein said dyes bind to a primary amine of a lysine residue of the protein and each said dye within said luminescent dyes carries a net +1 charge.
 3. The method of claim 1 wherein said set of matched luminescent dyes are cyanine dyes having the following structure:

wherein the dotted lines each represent carbon atoms necessary for the formation of one to three fused rings having five to six atoms in each ring, X and Y are selected from the group consisting of S, O and CH₃—C—CH₃, m is an integer from 1 to 3, one of R₁ and R₂ is a reactive group and the other is an alkyl.
 4. The method of claim 1 wherein each dye has at least one reactive group selected from the group consisting of isothiocyanate, isocyanate, N-hydroxysuccinimidyl ester, imido ester, glyoxal, carboxylic acid, haloacetamide, maleimide, alkyl halide, acid halide, azide, hydrazide, hydrazine, ketone and amino.
 5. The method of claim 1 wherein said set of matched luminescent dyes are neutral dyes bound to the primary amino of a lysine residue in the protein by a positively charged linker group.
 6. The method of claim 1 wherein said set of matched luminescent dyes are neutral dyes bound to a sulfhydryl group in the protein.
 7. The method of claim 1 wherein said set of matched luminescent dyes are derivatives of dipyrromethene boron difluoride dyes.
 8. The method of claim 1 wherein separating the dye-labeled proteins is by an electrophoretic method.
 9. The method of claim 8 wherein the electrophoretic method comprises one dimensional gel electrophoresis, two dimensional gel electrophoresis, capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, isotacophoresis, or micellar electrokinetic chromatography.
 10. The method of claim 1 wherein separating the dye-labeled proteins is by a chromatographic method.
 11. The method of claim 10 wherein the chromatographic method comprises affinity chromatography, size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography or ion exchange chromatography.
 12. The method of claim 1 further comprising quenching the reaction between the proteins and the dyes prior to mixing said dye labeled proteins.
 13. The method of claim 1 wherein the step of detecting differences in emitted color is by fluorescent microscopy.
 14. The method of claim 1 wherein the step of detecting differences in emitted color is by electronic imaging.
 15. The method of claim 1 wherein the proteins have binding sites for covalent binding to said dyes selected from the group consisting of lysine, carboxylic acid and sulfhydryl groups.
 16. The method of claim 1 wherein the proteins include glycoproteins having terminal sugar groups and preparing the extract of proteins further comprises the step of oxidizing terminal sugar groups to form aldehyde groups; and each dye of said set has a reactive group capable of forming a covalent bond with the aldehyde group.
 17. The method of claim 1 wherein the proteins include phosphoproteins.
 18. The method of claim 1 wherein preparing the extract of proteins further comprises digesting at least a portion of the proteins with enzyme for generating peptides.
 19. A method of comparing proteins of interest between at least two different samples comprising: (a) preparing a mixture of proteins from each of said at least two samples; (b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to said proteins within said mixture of proteins, wherein each dye within said set (1) has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal; (c) reacting each said mixture of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins; (d) mixing each of said dye labeled proteins to form a combined single mixture of different dye-labeled proteins; (e) separating the different dye-labeled proteins of interest within said combined mixture; and (f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection.
 20. The method of claim 19 wherein said set of matched luminescent dyes are cyanine dyes having the following structure:

wherein the dotted lines each represent carbon atoms necessary for the formation of one to three fused rings having five to six atoms in each ring, X and Y are each selected from the group consisting of O, S and CH₃—C—CH₃, m is an integer from 1 to 3, one of R₁ and R₂ is a reactive group and the other is an alkyl.
 21. The method of claim 19 wherein each dye has at least one reactive group selected from the group consisting of isothiocyanate, isocyanate, N-hydroxysuccinimidyl ester, imido ester, glyoxal, carboxylic acid, haloacetamide, maleimide, alkyl halide, acid halide, azide, hydrazide, hydrazine, ketone and amino.
 22. The method of claim 19 wherein the proteins have binding sites selected from the group consisting of lysine, carboxylic acid and sulfhydryl groups.
 23. The method of claim 19 wherein separating the dye-labeled proteins is by an electrophoretic method.
 24. The method of claim 23 wherein the electrophoretic method comprises one dimensional gel electrophoresis, two dimensional gel electrophoresis, capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, isotacophoresis, or micellar electrokinetic chromatography.
 25. The method of claim 19 wherein separating the dye-labeled proteins is by a chromatographic method.
 26. The method of claim 25 wherein the chromatographic method comprises affinity chromatography, size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography or ion exchange chromatography.
 27. The method of claim 19 wherein the proteins are glycoproteins having terminal sugar groups and preparing the extract of proteins further comprises the step of oxidizing terminal sugar groups to form aldehyde groups; and each dye of said set has a reactive group capable of forming a covalent bond with the aldehyde group.
 28. The method of claim 19 wherein the proteins are phosphoproteins and the dye includes an imidazole group, said method further comprising adding carbodiimide to said mixture of proteins and dyes for the reaction of step (c).
 29. The method of claim 19 wherein preparing the extract of proteins further comprises digesting at least a portion of the proteins with enzyme for generating peptides.
 30. A method of comparing proteins of interest between at least two different samples comprising: (a) preparing a mixture of proteins from each of said at least two samples; (b) providing a set of matched luminescent dyes chosen from a single class of dyes capable of covalently binding to proteins within said mixture of proteins, wherein each dye within said set (1) has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal; (c) reacting each mixture of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins; (d) mixing each of said dye labeled proteins to form a combined single mixture of different dye-labeled proteins; (e) separating different dye-labeled proteins of interest within said combined mixture; and (f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection.
 31. The method of claim 30 wherein separating the dye-labeled proteins is by an electrophoretic method.
 32. The method of claim 31 wherein the electrophoretic method comprises one dimensional gel electrophoresis, two dimensional gel electrophoresis, capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, isotacophoresis, or micellar electrokinetic chromatography.
 33. The method of claim 30 wherein separating the dye-labeled proteins is by a chromatographic method.
 34. The method of claim 33 wherein the chromatographic method comprises affinity chromatography, size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography or ion exchange chromatography.
 35. The method of claim 30 wherein the proteins are glycoproteins having terminal sugar groups and preparing the extract of proteins further comprises the step of oxidizing terminal sugar groups to form aldehyde groups; and each dye of said set has a reactive group capable of forming a covalent bond with the aldehyde group.
 36. The method of claim 30 wherein preparing the extract of proteins further comprises digesting the proteins with enzyme for generating peptides.
 37. The method of claim 30 wherein the proteins are phosphoproteins and the dye includes an imidazole group, said method further comprising adding carbodiimide to said mixture of proteins and dyes for the reaction of step (c).
 38. The method of claim 30 wherein each dye has at least one reactive group selected from the group consisting of isothiocyanate, isocyanate, N-hydroxysuccinimidyl ester, imido ester, glyoxal, carboxylic acid, haloacetamide, maleimide, alkyl halide, acid halide, azide, hydrazide, hydrazine, ketone and amino.
 39. A method of comparing protein compositions between at least two different cell samples comprising: (a) preparing an extract of proteins from each of said at least two cell samples; (b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, and, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal; (c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins; (d) mixing said dye labeled proteins to form a single mixture of different dye-labeled proteins; (e) separating the dye-labeled proteins within said mixture by a chromatographic method; and (f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection. 